! PROJECT SUMMARY Chlamydia trachomatis is the leading cause of human sexually transmitted infections of bacterial origin world- wide. Infections are often asymptomatic and, if left untreated, have long-term consequences on female repro- ductive health. Although treatable with antibiotics, reinfection rates are high due the lack of long-term protective immunity from infection, and vaccines are not available. Over the years, data collected from human epidemio- logical studies, murine models of infection and infected cancer-derived epithelial cells, such as HeLa cells, have contributed to a better understanding of the infection process. However, there are substantial limitations with the current models available, due to accessibility to or tractability of the samples (human and mice) or physiological relevance (HeLa cells). Over the past decade, 3D organoids, derived from adult or pluripotent stem cells, have been generated from various tissues, including the female reproductive tract, and shown to closely recapitulate the cellular composition, and biology/functionality of the original organs' epithelium. There- fore, organoids offer a unique model system to study host-pathogen interaction in a relatively simple, yet rele- vant ex vivo environment. Here, we propose to develop an endometrial organoid (EMO) - C. trachomatis model of infection. Our preliminary data indicate that C. trachomatis can infect and undergo a productive developmen- tal cycle in EMO. We now want to further characterize C. trachomatis infection in EMO. Our hypothesis is that key features of Chlamydia developmental cycle will be recapitulated during EMO infection. In addition, we want to develop methods to manipulate EMO to address the role of specific host factors in infection. Altogether, pio- neering Chlamydia infection of 3D EMO will be instrumental in investigating the infection process of this medi- cally important bacterial pathogen, in a relevant ex vivo model system. This model will provide a better appre- ciation of the molecular fundamentals of Chlamydia infection, which could help in the design of efficient strate- gies to combat and prevent the disease.